Small Hero with Great Powers: Vaccinia Virus E3 Protein and Evasion of the Type I IFN Response

Poxviridae have developed a plethora of strategies to evade innate and adaptive immunity. In this review, we focused on the vaccinia virus E3 protein, encoded by the E3L gene. E3 is present within the Chordopoxvirinae subfamily (with the exception of the avipoxviruses and molluscum contagiosum virus) and displays pleiotropic effects on the innate immune system. Initial studies identified E3 as a double-stranded RNA (dsRNA)-binding protein (through its C terminus), able to inhibit the activation of protein kinase dependent on RNA (PKR) and the 2′5′-oligoadenylate synthetase (OAS)/RNase L pathway, rendering E3 a protein counteracting the type I interferon (IFN) system. In recent years, N-terminal mutants of E3 unable to bind to Z-form nucleic acids have been shown to induce the cellular death pathway necroptosis. This pathway was dependent on host IFN-inducible Z-DNA-binding protein 1 (ZBP1); full-length E3 is able to inhibit ZBP1-mediated necroptosis. Binding to what was identified as Z-RNA has emerged as a novel mechanism of counteracting the type I IFN system and has broadened our understanding of innate immunity against viral infections. This article gives an overview of the studies leading to our understanding of the vaccinia virus E3 protein function and its involvement in viral pathogenesis. Furthermore, a short summary of other viral systems is provided.


Introduction
The family of Poxviridae encompasses a group of large (200 nm × 300 nm), brickshaped, enveloped viruses that are known to infect both invertebrates (subfamily Entomopoxvirinae) and vertebrates (subfamily Chordopoxvirinae) [1][2][3]. Poxviruses replicate entirely in the cytoplasm, which distinguishes them from most other DNA viruses. The poxviral genome is a single, linear, double-stranded DNA (dsDNA) molecule, which ranges in size from 130 to 300 kbp [4]. In its conserved central region, the poxviral genome encodes proteins essential for virus replication, such as transcription and DNA replication enzymes. The more variable terminal regions are known to encode non-essential virulence factors, proteins affecting host range, and proteins involved in modulating the host's innate immune response [4][5][6].
The most studied member of the Poxviridae family is vaccinia virus (VACV), which is the live vaccine used to immunize against and eradicate smallpox [7]. Vaccinia virus belongs to the orthopoxvirus genus, which encompasses other notable members: for instance, variola virus (the causative agent of human smallpox), monkeypox virus, and cowpox virus (known human and animal pathogens) [5,7,8]. During poxviral infection, transcription of the viral genome results in the production of dsRNA and Z-RNA. Furthermore, dsRNA is sensed by cytosolic sensors RIG-I/MDA5 and cGAS, which induce a signaling cascade leading to the production of proinflammatory cytokines and type I IFN. IFN is sensed by neighboring cells, where it induces the expression of ISGs, including OAS, PKR, and ZBP1. Activation of OAS and PKR by dsRNA leads to RNA degradation and inhibition of translation, respectively. Activation of ZBP1 by Z-RNA results in induction of cellular death pathway necroptosis. The VACV E3 protein binds to dsRNA and thereby inhibits its recognition by RIG-I/MDA5 and cGAS (red text and red lines). E3 also inhibits activation of OAS and PKR by binding to dsRNA, and inhibits activation of ZBP1 by binding to zRNA. Abbreviations used in this figure include: eIF2α: eukaryotic translation initiation factor 2 subunit alpha; IFN: interferon; IFNAR1/2: IFN alpha and beta receptor subunit 1/2; IκBα: NFκB inhibitor alpha; IKKα/β/ε: IκBα kinase alpha/beta/epsilon; IRF3/7/9: interferon regulatory factor 3/7/9; ISG: IFN-stimulated gene; ISRE: IFNsensitive response element; JAK1: Janus kinase 1; MAVS: mitochondrial antiviral-signaling protein; MDA5: melanoma differentiation-associated protein 5; MLKL: mixed-lineage kinase-like; NEMO: NFκB essential modulator; NFκB: nuclear factor of kappa-light-chain-enhancer of activated B-cells; Figure 1. Summary of type I IFN response induction by poxviruses and its inhibition by VACV E3 protein. During poxviral infection, transcription of the viral genome results in the production of dsRNA and Z-RNA. Furthermore, dsRNA is sensed by cytosolic sensors RIG-I/MDA5 and cGAS, which induce a signaling cascade leading to the production of proinflammatory cytokines and type I IFN. IFN is sensed by neighboring cells, where it induces the expression of ISGs, including OAS, PKR, and ZBP1. Activation of OAS and PKR by dsRNA leads to RNA degradation and inhibition of translation, respectively. Activation of ZBP1 by Z-RNA results in induction of cellular death pathway necroptosis. The VACV E3 protein binds to dsRNA and thereby inhibits its recognition by RIG-I/MDA5 and cGAS (red text and red lines). E3 also inhibits activation of OAS and PKR by binding to dsRNA, and inhibits activation of ZBP1 by binding to zRNA. Abbreviations used in this figure include: eIF2α: eukaryotic translation initiation factor 2 subunit alpha; IFN: interferon; IFNAR1/2: IFN alpha and beta receptor subunit 1/2; IκBα: NFκB inhibitor alpha; IKKα/β/ε: IκBα kinase alpha/beta/epsilon; IRF3/7/9: interferon regulatory factor 3/7/9; ISG: IFN-stimulated gene; ISRE: IFN-sensitive response element; JAK1: Janus kinase 1; MAVS: mitochondrial antiviral-signaling protein; MDA5: melanoma differentiation-associated protein 5; MLKL: mixed-lineage kinase-like; NEMO: NFκB essential modulator; NFκB: nuclear factor of kappa-light-chain-enhancer of activated B-cells; OAS: oligoadenylate synthetase; PKR: protein kinase dependent on RNA; RIG-I: retinoic acid-inducible gene I; RIPK3: receptor-interacting protein kinase 3; STAT1/2: signal transducer and activator of transcription 1/2; STING: stimulator of interferon genes; TRAF3/6: tumor necrosis factor receptor-associated factor 3/6; TBK1: TRAF family member-associated NFκB activator (TANK)binding kinase 1; TYK2: tyrosine kinase 2; ZBP1: Z-DNA-binding protein 1. There are several mechanisms by which poxviruses are able to inhibit the type I IFN response. They are reviewed in [61]. This review focuses on vaccinia virus E3 protein and a novel way of affecting immune response by inhibition of necroptosis, which is dependent on the IFN-inducible ZBP1 protein. We provide an historical overview of how E3 protein was discovered and outline the progress during which E3 function was discerned.

Discovery of E3 Protein
Very early on in the study of the interferon system, it became clear that viruses differed in their sensitivity to interferon. Vaccinia virus (VACV) was shown by the Youngner group to be quite resistant to the pre-treatment of cells with type I IFN [62]. Not only was VACV IFN R , but it rescued both vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) from the effects of IFN [9,62,63]. For rescue of EMCV, rescue correlated with inhibition of PKR activation by VACV [9].
Whitaker-Dowling first demonstrated that VACV encoded an inhibitor of the IFNinducible antiviral protein kinase, PKR [64]. Our group purified this PKR-inhibitor from infected cells, obtained protein sequence information for the putative inhibitor, and identified it as the product of the VACV E3L gene [65]. Isolated E3 protein had potent PKRinhibitory activity, and VACV deleted of E3L (vp1080) failed to inhibit PKR activation (Figures 1 and 2) [65,66]. Unlike wt VACV, VACV vp1080 failed to replicate in most cell lines in culture and was IFN S in rabbit RK-13 cells where it did replicate [67,68]. VACV vp1080 failed to rescue VSV from the antiviral effects of IFN, but still efficiently rescued EMCV, suggesting the presence of a second IFN R gene [69]. This was identified as K3L, which encodes an eIF2α homologue, which could inhibit PKR or any of the other eIF2α protein kinases [70,71]. K3L partially rescued EMCV from the effects of IFN but had no effect on IFN S of VSV [69]. Likewise, VACV deleted of K3L (vp872) failed to rescue EMCV, but efficiently rescued VSV from the effects of IFN [69]. Thus, E3L was essential for the rescue of VSV, but dispensable for the rescue of EMCV, while K3L led to at least partial rescue of EMCV, but had no effect on the rescue of VSV from the effects of IFN, suggesting a complementary function of the two proteins against the host's IFN system.

C Terminus of E3 Contains a dsRNA-Binding Domain
When E3L was first identified as the primary IFN R gene of vaccinia virus, a BLAST search identified the C-terminal half of the protein as having sequence similarity with proteins known to bind to dsRNA, such as PKR and RNase III [66]. We were able to show that E3L-encoded proteins bound specifically to dsRNA, but not single-stranded RNA (ssRNA) or dsDNA, and that this conserved domain was necessary and sufficient for binding ( Figure 2 and Table 1) [72]. IFN R and the broad host range of VACV also mapped to this dsRNA binding motif. Furthermore, a virus missing the entire N-terminal half of E3L (VACV E3L∆83N) was IFN R in RK-13 cells and replicated in a wide variety of human and monkey cells [73,74] despite leading to the activation of PKR and OAS, albeit at very late times post-infection. Surprisingly, both the C-terminal dsRNA-binding domain, which is essential for E3 function and the N-terminal domain, which is dispensable for IFN R and a broad host range, are conserved amongst the majority of poxviruses that infect mammals [75]. Furthermore, the distantly related ORF virus E3L homologue could fully replace VACV E3L for IFN R and replication in most cells in culture [74]. This shows that while we could not detect an essential function for the N-terminal domain in rabbit, human, or monkey cells in culture, it was in fact conserved amongst poxviruses, suggesting an important role in vivo. While the N-terminal domain of E3 was conserved amongst most mammalian poxviruses, it showed no homology with other proteins in available databases. vp1080 failed to rescue VSV from the antiviral effects of IFN, but still efficiently rescued EMCV, suggesting the presence of a second IFN R gene [69]. This was identified as K3L, which encodes an eIF2α homologue, which could inhibit PKR or any of the other eIF2α protein kinases [70,71]. K3L partially rescued EMCV from the effects of IFN but had no effect on IFN S of VSV [69]. Likewise, VACV deleted of K3L (vp872) failed to rescue EMCV, but efficiently rescued VSV from the effects of IFN [69]. Thus, E3L was essential for the rescue of VSV, but dispensable for the rescue of EMCV, while K3L led to at least partial rescue of EMCV, but had no effect on the rescue of VSV from the effects of IFN, suggesting a complementary function of the two proteins against the host's IFN system.

E3 Protein Is VACV Virulence Factor
Since the N-terminal domain of E3 was dispensable for IFN R in RK-13 cells and for replication in a wide variety of human and monkey cells, despite being evolutionarily conserved, we decided to ask if this domain was necessary for pathogenesis in the mouse model. VACV WR is a mouse-adapted VACV strain that has an intranasal (IN) LD 50 of 10 3-10 4 pfu, depending on the strain of mouse [76,77]. VACV WR ∆E3L is highly attenuated in the mouse model, having an LD 50 greater than 10 7 pfu. Both the C-terminal dsRNAbinding domain and the conserved N-terminal domain are necessary for pathogenesis since both VACV WR E3L∆83N and VACV WR E3L∆26C (which has no detectable affinity for dsRNA) are highly attenuated (Table 1) [76]. This was the first suggestion that the conserved N-terminus of E3L was biologically relevant.

E3 Protein Is a Type I IFN Antagonist
To begin to understand the function of E3 in vivo, we infected IFNAR −/− mice with VACV WR ∆E3L and VACV WR E3L∆83N. While the lack of an IFN signaling system did not restore pathogenesis to VACV WR ∆E3L when animals were infected IN, it partially restored pathogenesis upon intracranial (IC) infection [77]. This is likely due to the lower dose of virus needed to induce pathogenesis when the virus is administered IC. Only partial restoration of pathogenesis in IFNAR −/− mice suggests that there is a non-IFN-inducible pathway that can at least partially inhibit pathogenesis induced by VACV WR ∆E3L. VACV WR ∆E3L also gains virulence in MDA5 −/− , MAVS −/− , and IRF3 −/− mice, consistent with a key role for the IFN system in blocking pathogenesis by VACV WR ∆E3L [78]. Pathogenesis of VACV WR E3L∆83N was fully restored in IFNAR −/− mice when the virus was administered either IN or IC [77]. This suggested that the sole function of the conserved N-terminal domain in vivo is to counteract the type I IFN system.

Discovery of Z-DNA
In the late 1990s, Alex Rich's lab screened a chicken cDNA library for proteins that could bind to Z-DNA [79]. They cloned out the IFN-inducible form of the host protein ADAR1. ADAR1 is an RNA modifying enzyme that can deaminate adenosine in dsRNA or RNA with a secondary structure [80,81]. The Rich lab mapped the domain that could bind Z-DNA (Zα domain) to an N-terminal region found only in the IFN-inducible p150 form of ADAR1 ( Figure 3) [82,83]. The constitutively expressed form of ADAR1, p110, is lacking this domain. Both p110 and p150 contain a second Zα-related domain (Zβ) that, however, has a key amino acid change that prevents interaction with Z-DNA [75,84].

The N Terminus of E3 Is a ZBD and Binding to a Z-NA Is Required for VACV Pathogenesis
A database search at the time showed a similarity between the Zα domain of ADAR1 to one other IFN-inducible protein, ZBP1-which at the time was known as DLM-1 and later renamed DAI (ZBP1 contains two Zα domains, Zα1 and Zα2)-and to the conserved N-terminal domain of poxvirus E3 protein, that we showed is essential for pathogenesis in mice ( Figure 3) [34,76,85,86]. This allowed us to collaborate with the Rich lab to ask if the capacity to bind to Z-DNA is critical for VACV to be pathogenic in mice. We first made chimeric viruses, replacing the N-terminal domain of E3 protein with either the Zα or Zβ domain of ADAR1, or the Zα1 domain of ZBP1 ( Figure 2). Replacing the N-terminus of E3 with either of the two Z-DNA binding domains-the Zα domain of ADAR1 or the Zα1 domain of ZBP1-led to viruses with virulence indistinguishable from wt VACV WR, while replacing the N-terminus of E3 with the related Zβ domain of ADAR1, which cannot bind Z-DNA, led to a virus that was attenuated to a similar degree as E3L∆83N virus, which completely lacks the conserved N-terminus (Table 1) [75]. This allowed us to perform a structure/function analysis based on the crystal structure of the Zα domain of ADAR1 bound to Z-DNA [87]. Mutation of residues that contacted Z-DNA in crystal structures led to proteins with reduced affinity for Z-DNA and led to a virus that was attenuated in vivo [75]. Furthermore, mutation of several residues not forming contacts with Z-DNA in the crystal structure had wt, nanomolar affinity for Z-DNA, and wt virulence in vivo. Equivalent mutations in authentic E3 protein led to viruses with equivalent virulence in vivo [75]. As mentioned above, the Zβ domain of ADAR1 cannot bind Z-DNA [84]. In the Zβ domain of ADAR1, residue 335, which in the equivalent position in Zα domains is a conserved tyrosine (Y177 in the Zα domain of ADAR1), has been changed to an isoleucine. Residue Y177 makes several key interactions with Z-DNA in the crystal structure and, when mutated, attenuates the virus. A mutation of isoleucine 335 to tyrosine (I335Y) partially restored binding to Z-DNA, and partially restored virulence in the virus expressing a chimeric ZβI335Y E3 protein [75]. Thus, all of our data is consistent with the binding of E3 protein to a Z-form nucleic acid in infected cells being critical for VACV pathogenesis in mice.
VACV WR ΔE3L and VACV WR E3LΔ83N. While the lack of an IFN signaling system did not restore pathogenesis to VACV WR ΔE3L when animals were infected IN, it partially restored pathogenesis upon intracranial (IC) infection [77]. This is likely due to the lower dose of virus needed to induce pathogenesis when the virus is administered IC. Only partial restoration of pathogenesis in IFNAR −/− mice suggests that there is a non-IFN-inducible pathway that can at least partially inhibit pathogenesis induced by VACV WR ΔE3L. VACV WR ΔE3L also gains virulence in MDA5 −/− , MAVS −/− , and IRF3 −/− mice, consistent with a key role for the IFN system in blocking pathogenesis by VACV WR ΔE3L [78]. Pathogenesis of VACV WR E3LΔ83N was fully restored in IFNAR −/− mice when the virus was administered either IN or IC [77]. This suggested that the sole function of the conserved N-terminal domain in vivo is to counteract the type I IFN system.

Discovery of Z-DNA
In the late 1990s, Alex Rich's lab screened a chicken cDNA library for proteins that could bind to Z-DNA [79]. They cloned out the IFN-inducible form of the host protein ADAR1. ADAR1 is an RNA modifying enzyme that can deaminate adenosine in dsRNA or RNA with a secondary structure [80,81]. The Rich lab mapped the domain that could bind Z-DNA (Zα domain) to an N-terminal region found only in the IFN-inducible p150 form of ADAR1 ( Figure 3) [82,83]. The constitutively expressed form of ADAR1, p110, is lacking this domain. Both p110 and p150 contain a second Zα-related domain (Zβ) that, however, has a key amino acid change that prevents interaction with Z-DNA [75,84].

N Terminus of E3 Is Required for Full PKR Inhibition in 129 MEFs
The identification of both a biological function in vivo for the N-terminus (IFN R ) and a biochemical function (binding to Z-DNA) led us to redouble efforts to find cells in which VACV E3L∆83N had a phenotype. We discovered three murine cells in culture in which VACV E3L∆83N had a phenotype: 129 MEFs, L929 cells, and JC cells. IFN sensitivity of VACV E3L∆83N in 129 MEFs did not correlate with the binding of E3 proteins to Z-DNA but instead correlated with the inability to fully sequester dsRNA [77]. In addition to not binding to Z-DNA, VACV E3L∆83N cannot fully inhibit PKR activation in cells in culture [26]. The residues necessary to fully inhibit PKR are different from the residues necessary for Z-DNA binding. A failure to fully inhibit PKR by N-terminal mutants of E3 is due to a failure to fully sequester dsRNA in infected cells [26,72,74]. In 129 MEFs, IFN R of VACV E3L∆83N could be fully recovered in PKR −/− cells or by PKR knock-down [77]. Additionally, selection of a virus that makes much less dsRNA than wt VACV fully rescued IFN R of an N-terminal mutant of E3 [88]. However, the pathogenesis of VACV E3L∆83N was not restored in PKR −/− mice [77]. This suggested that an additional IFN-inducible function, perhaps in addition to activation of PKR, is important for the attenuation of VACV E3L∆83N in vivo.

Binding to Z-NAs by E3 Is Essential for VACV IFN Resistance
In mouse L929 cells, VACV E3L∆83N is IFN S while wt VACV is IFN R , and in mouse JC cells VACV E3L∆83N fails to replicate, while wt VACV replicates efficiently (see Figure 4) [40,88]. Unlike replication in 129 MEFs, IFN R in L929 cells and replication in JC cells correlate with Z-DNA binding among mutants of E3L (Figure 4) [16,88]. Thus, the phenotype in N-terminal mutants of E3 in these cells correlates with the phenotype seen in vivo (see Section 4.2 and Table 1). In mouse L929 cells, VACV E3LΔ83N is IFN S while wt VACV is IFN R , and in mouse JC cells VACV E3LΔ83N fails to replicate, while wt VACV replicates efficiently (see Figure  4) [40,88]. Unlike replication in 129 MEFs, IFN R in L929 cells and replication in JC cells correlate with Z-DNA binding among mutants of E3L (Figure 4) [16,88]. Thus, the phenotype in N-terminal mutants of E3 in these cells correlates with the phenotype seen in vivo (see Section 4.2 and Table 1).

Necroptosis Inhibition Is Conferred by N Terminus of E3
In mouse L929 cells, the infection of IFN-treated cells with VACV E3LΔ83N leads to rapid explosive cell death, by 6 h post-infection. Treatment with a pancaspase inhibitor, Z-VAD-FMK (zVAD), did not rescue either cell death or IFN R of VACV E3LΔ83N [40]. This suggested that cell death is caspase-independent and thus, likely not apoptosis or pyroptosis [89]. Necroptosis, which is a caspase-independent form of programmed cell death, requires the cellular kinase RIPK3 to phosphorylate MLKL, the executioner of necroptotic cell death, on Ser358 (Ser345 in mice) [55,[90][91][92][93]. RIPK3 kinase activity can be inhibited by the small molecule GSK872. GSK872 treatment inhibits VACV E3LΔ83N-induced cell death and fully rescues VACV E3LΔ83N from the effects of IFN-treatment [40]. VACV E3LΔ83N infection of IFN-treated L929 cells leads to phosphorylation of MLKL that is inhibitable by GSK872 [40]. Phosphorylated MLKL can migrate to the plasma membrane, trimerize and form pores in the membrane that allow fluid influx, swelling and bursting [59,60,94,95]. MLKL trimerizes in VACV E3LΔ83N-infected cells and trimerization is blocked by GSK872 treatment [40]. CRISPR knockout of MLKL fully rescues VACV

The ZBD of E3 Interacts with Z-RNA to Inhibit Necroptosis
Data demonstrating sensing of VACV E3L∆83N infection by ZBP1 suggests that the Zα domains of E3 and ZBP1 may be competing with each other for binding to a Z-form nucleic acid in infected cells. Furthermore, Z-form NA can be detected by immunofluorescence (IF) in VACV-infected but not uninfected cells [16]. By IF, Z-NA levels were highest in VACV E3L∆E3L-infected cells, followed by VACV E3L∆83N with the lowest amounts in wt VACV-infected cells. Z-NA staining was abolished by pre-treatment with RNase, but not DNase, suggesting that Z-RNA is present in VACV-infected cells [16]. This suggests that a functional N-terminus may be sequestering Z-RNA. This was confirmed by flow cytometry, where Z-RNA was detectable in VACV E3L∆83N, but not wt VACV-infected cells. Over-expression of ZBP1 masked Z-RNA detection in VACV E3L∆83N-infected cells, which was consistent with ZBP1 and the Zα domain of E3 competing for binding to the same Z-RNA [16]. This is also consistent with our data in vivo with chimeric viruses, where the Zα domain of ADAR1 or the Zα1 domain of ZBP1 could functionally replace the Zα domain of E3 protein [75]. We have recently shown that the Zα domain of ADAR1 and the Zα1 or Zα2 domain of ZBP1 ( Figure 3) can functionally replace the Zα domain of E3 protein in L929 cells in culture, preventing sensing of VACV infection by ZBP1 [16].

ZBP1-Mediated Necroptosis Underlies Attenuation of VACV E3L∆83N In Vivo
To ask if the role that we have seen in necroptosis in cells in culture correlates with the role of the N-terminus in pathogenesis in mice, we have evaluated the pathogenesis of VACV E3L∆83N in ZBP1 −/− and RIPK3 −/− mice. In wt mice infected IN, VACV E3L∆83N replicates to a high titer in the nose, but spreads poorly or replicates poorly at distal sites, including the lung, brain, and spleen. The spread is rescued to a large extent in IFNAR −/− mice [77]. In wt mice, VACV E3L∆83N is attenuated by at least four logs on IN infection, and virulence, too, is rescued in IFNAR −/− mice [77]. Consistently, VACV E3L∆83N is further attenuated by IFN treatment of wt mice prior to infection ( Figure 5A). To a large extent, attenuation, the spread to distal organs, and IFN resistance is rescued in both ZBP1 −/− and RIPK3 −/− mice, indicating that much of the IFN sensitivity of VACV E3L∆83N in vivo is likely due to induction of the ZBP1/RIPK3 axis resulting in necroptosis ( Figure 5A,B) [40]. Furthermore, VACV E3L P63A, a point mutant unable to bind to Z-NA and known not to inhibit necroptosis in cells in culture (Table 1), displays decreased pathogenicity in wt mice, but its pathogenicity is rescued in ZBP1 −/− and RIPK3 −/− mice ( Figure 5C). Furthermore, we demonstrated that infection of wt mice IN with VACV E3L∆83N resulted in the induction of necroptosis, which was not observed in mice infected with wt VACV or uninfected mice ( Figure 5D) [101].
Biomedicines 2022, 10, x FOR PEER REVIEW 11 of 19 resulted in the induction of necroptosis, which was not observed in mice infected with wt VACV or uninfected mice ( Figure 5D) [101].

Cytomegaloviruses
One of the first insights into necroptosis being involved in antiviral immunity comes from studies done by Ed Mocarksi's group showing that murine cytomegalovirus (MCMV)encoded M45 protein had the ability to block RIPK3-mediated necroptosis [102]. Infection of cells with MCMV-M45mutRHIM mutant led to induction of necroptosis, which was independent of RIPK1 as necrostatin-1 (RIPK1 inhibitor) and RIPK1-specific shRNAs had no effect on cell death induced by the mutant virus [103]. MCMV-M45mutRHIM induced necroptosis only in cells expressing ZBP1, which was the first time ZBP1 was implicated in this cell death pathway [44]. Furthermore, MCMV-M45mutRHIM is attenuated in wt mice, but not in RIPK3 −/− or ZBP1 −/− mice [44,103]. These studies demonstrate that modulation of necroptosis is crucial for successful viral pathogenesis, which remains consistent with what we see for VACV E3L∆83N (Table 1) [40].
Maelfait and colleagues established the importance of binding to Z-NAs for necroptosis induced during MCMV infection. MCMV-M45mutRHIM was only able to induce necroptosis in the presence of wt ZBP1, but not when Zα1 and Zα2 domains of ZBP1 were mutated [42]. Furthermore, MCMV-M45mutRHIM infection is successfully cleared in wt mice, but not in knock-in mice expressing ZBP1 with mutated Zα domains, suggesting a role for Z-NAs in clearing MCMV infection. Moreso, ZBP1 was found associated with RNA during MCMV-M45mutRHIM infection and induction of cell death required RNA synthesis but not viral DNA replication, implicating a role for Z-RNA in virally induced necroptosis, similarly to what we suggest for VACV E3L∆83N [16,42]. For both viruses, these Z-RNAs have been shown to be of viral origin [16,104]. Interestingly, human cytomegalovirus (HCMV) displays species-dependent regulation of the necroptosis pathway. HCMV protein UL36 binds to MLKL, leading to MLKL degradation and inhibition of necroptosis in human cells, but not in murine cells (Muscolino et al. 2021).

Herpes Simplex Virus 1
Aside from cytomegaloviruses, other herpesviruses are known to modulate necroptosis, and this modulation is species-dependent. Human herpes simplex virus 1 (HSV-1) infection is successfully cleared in wt mice but not in RIPK3 −/− mice [56]. In murine cells, HSV-1 induces necroptosis, which is independent of ZBP1 but depends on viral ribonucleotide reductase large subunit (IC6), which interacts in an RHIM-mediated manner with RIPK1 and RIPK3. IC6 facilitates and is required for RIPK1-RIPK3 and RIPK3-RIPK3 interaction and induction of necroptosis in mice [56,105]. In contrast, IC6 has been shown to inhibit necroptosis in human cells by impairing RIPK1-RIPK3 necrosome formation and reduction of RIPK1 autophosphorylation, suggesting that HSV-1 evolved to escape necroptosis in humans for the sake of establishing a successful infection [106][107][108]. Interestingly, when the RHIM of IC6 is mutated, HSV-1 is still able to induce necroptosis in mice, which this time is dependent on ZBP1. IC6mutRHIM virus infection is also cleared in wt mice, but not in ZBP1 −/− mice [109]. Strikingly, ZBP1-mediated necroptosis is also induced in human cells infected with IC6mutRHIM virus, showing that the ZBP1-mediated pathway is species-independent [109].

Influenza A Virus
Z-NAs have been shown to play a role in the pathogenesis of RNA viruses as well. Influenza A virus (IVA) infection is sensed by ZBP1, which activates RIPK3 and triggers MLKL-mediated necroptosis and FADD-mediated apoptosis [43,110]. ZBP1 senses viral ribonucleoprotein (vRNP) complexes, including IAV genomic RNA, nucleoprotein (NP), and polymerase subunit (PB1), and cells lacking ZBP1 or expressing Zα mutants of ZBP1 fail to trigger IAV-induced necroptosis or apoptosis, indicating that viral genomic Z-RNA plays a role [41,43,111]. ZBP1-deficient mice succumb to uncontrolled IAV infection [43]. In other studies, ZBP1 −/− and MLKL −/− mice displayed reduced inflammatory response and epithelial damage, demonstrating the pro-inflammatory nature of the necroptotic cell death [41,112]. Interestingly, ZBP1 sensing of viral Z-RNA takes place in the nucleus and it requires ZBP1 ubiquitination induced by IAV infection [111,112].

Conclusions and Outlook
Vaccinia virus E3 protein is an immune evasion protein conserved among poxviruses [72,75]. E3 displays a pleiotropic effect, with an ability to counteract multiple arms of the host innate immunity: PKR, OAS/RNase L, STING, RIG-I/MDA5, RNA Pol III, TLR2/-8/-9 (described in [61]). With the development of our understanding of PCD pathways, a new function for E3 protein has been revealed as an inhibitor of necroptotic cell death. Additionally, the function of E3 ZBD has been characterized in more detail and added to the progress in understanding the biological function of Z-NAs and the relevant associated proteins, such as ZBP1. Studies described here shed light on the biological significance of Z-NAs, demonstrating their involvement in viral pathogenesis and host innate immunity. The same studies also showed how viruses have evolved to counteract these Z-NA-mediated responses in order to evade host immunity.
VACV E3 protein is the first case of a poxviral protein that has been shown to bind to Z-RNA and inhibit necroptosis ( Figure 1). More studies are needed to show if other proteins with similar abilities are present in the poxviral family and other viral families. There are still a few unknown aspects of the necroptosis pathway triggered by Z-RNA that need further investigation. It remains unknown what kind of RNA is responsible for necroptosis induction during VACV E3L∆83N infection. It is unclear whether it is an RNA of a specific gene or any RNA that takes on the Z-form. Furthermore, the mechanism of Z-formation needs to be examined. Poxviruses are known to induce oxidative stress, which is known to lead to damage in nucleic acids by the introduction of the hydroxyl group into nucleotide structure (for instance, formation of 8-oxoguanine from guanine), which renders such nucleic acid more prone to flip to the Z-form [113][114][115]. Oxidative stress that is induced during poxviral infection could lead to the formation of Z-RNA, which subsequently could lead to the activation of necroptosis. Finally, it will be exciting to see how studies described in this review will contribute to the development of novel therapeutic approaches based on Z-RNA to enhance type I IFN immunity.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.